Specific Aims The major goal of this proposal is to understand mechanisms by which astrocytes interact with neurons to modulate neural circuit activity and animal behavior. Many studies over the past two decades have suggested that astrocytes respond directly to neurotransmission and in turn signal back to neurons to regulate circuit activity. Definitive in vivo examples have remained elusive, but if astrocytes indeed receive synaptic information and feedback to modulate neuronal activity it is imperative that we identify and define these mechanisms-astrocyte control of neuronal activity could represent a previously unappreciated but prominent mechanism for regulating information processing in the brain and animal behavior. We recently made the exciting observation that in live preparations Drosophila astrocytes exhibit rhythmic fluctuations in intracellular Ca2+ similar to those observed in astrocytes of awake behaving mice. Astrocyte Ca2+ signaling in larvae is not altered by application of glutamate, GABA, or acetylcholine, but is potently stimulated by octopamine (OA), the invertebrate equivalent of norepinephrine (NE). Intriguingly, NE was recently found to stimulate astrocytic Ca2+ signaling in mice, although the NE site of action (e.g. neurons, astrocytes, or both) remains unclear, molecules required in astrocytes for NE-induced Ca2+ signals have not been identified, and their physiological roles remain enigmatic. Furthermore, using forward genetic approaches we identified the G protein-coupled octopamine-tyramine receptor (Oct-tyrR) and the TRP channel Waterwitch (Wtrw) as novel, astrocyte-expressed molecules required for activation of OA-induced astrocyte Ca2+ transients. Remarkably, depletion of Wtrw from astrocytes leads to defects in both olfaction and touch- induced startle responses, implicating Wtrw and astrocyte Ca2+ signaling in animal behavior. Our identification of Oct-tyrR and Wtrw as novel critical regulators of astrocyte Ca2+ signaling opens the door to an exploration of the functional significance astrocyte Ca2+ signaling in vivo, which is proposed herein. Our work will define new components of the astrocyte Ca2+ signaling machinery and their importance in astrocyte-neuron signaling events (Aim 1); determine how astrocyte Ca2+ signaling events are regulated by neural circuits and how they reciprocally modulate neuronal activity in vivo (Aim 2); and explore the role of astrocyte Ca2+ signaling molecules in simple sensory-driven behaviors, circadian and arousal behaviors, and a touch induced startle response.